Note: Descriptions are shown in the official language in which they were submitted.
CA 02212832 1997-08-13
WO 96(26269 PCT~G1~96/00423
T ~ OMRIN PREPP~U~T~ON
Field of the Invention
The present invention relates to a novel process for
the production of thrombin particularly human thrombin
and to thrombin preparations capable of being produced in
a freeze dried form, which may be heat-treated in order
to inactivate any viruses present.
Background of the Invention
Thrombin is the product of the activation of
prothrombin by Factor Xa in plasma. It is a potent
broadly specific serine proteinase that converts
fibrinogen to fibrin and promotes fibrin cross-linking by
activating Factor XIII. Amongst a number of other
observed biological activities, thrombin also controls
several feedback loops in the clotting cascade and
induces the platelet release reaction (1, 2)
Thrombin has been used as a topical haemostatic
agent for many years. However, it is as a component of
fibrin sealant (fibrin glue) that the clinical use of
thrombin is likely to expand. Thrombin is used in fibrin
sealant to convert fibrinogen to fibrin on a cut surface
or within a graft and numerous surgical applications have
been described in a wide range of surgical specialities
(3, 4).
Bovine thrombin is currently used widely as a
topical haemostatic agent or as a component of commercial
fibrin sealant products. While such thrombin products
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W 096/26269 PCT/GB96/00423
are biologically effective, they are associated with
well-documented risk of allergic responses and induction
of antibodies to the bovine thrombin or to impurities
such as bovine factor V, usually after repeat use (5, 6
and 7). Ortel et al (8) recently concluded that such
acquired coagulation factor inhibitors probably occur
more commonly than is currently appreciated and although
frequently clinically benign, these inhibitors may be
associated with life-threatening haemorrhage. For this
reason the development of a process to produce human
thrombin suitable for use as a topical haemostat or for
inclusion in a fibrin sealant product, has been sought.
Intrinsically, thrombin is formed when prothrombin
(Factor II) is converted by activated Factor X, activated
Factor V, phospholipid and calcium ions into thrombin.
Conversion of prothrombin to thrombin can occur without
some of the associated components, however, the rate of
conversion is undesirably slow.
There are three main in vitro prothrombin conversion
methods known in the art. The first method relies on the
use of thromboplastin. Prothrombin is converted to
thrombin using thromboplastin preferably in the presence
of calcium chloride. This is described in a number of
patent specifications such as EP 0439156A and EP
0505604A. A disadvantage of this method is that the
thromboplastin is usually a crude preparation which has
been prepared from freshly homogenised brain, lung or
intestinal tissue. This procedure is not appropriate for
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W 0~6126269 PCT/GB96100423
the preparation of human thrombin as the reagents,
depending on their source, can carry the risk of virus or
cross-species cont~m;n~tion.
A second method utilises some components of snake
venom to yield thrombin (9, 10, 11). However, it has
been reported that some of the venoms do not cleave the
same bonds within prothrombin, as the natural activator,
Factor Xa (12). Thus, there may be dangerous
implications should a non-physiological form of thrombin
be used clinically.
The third in vitro method is essentially the same as
the intrinsic in vivo process, wherein prothrombin is
converted to thrombin by activated Factor X, Factor V,
phospholipid and calcium ions under near physiological
conditions. This has been described, for instance in, EP
0528701, EP 0378798 and US 5,219,995. However, the
thrombin produced is often unstable unless exogenous
proteins, polyols and/or sugars are added to the thrombin
to stabilise it.
Since human thrombin is derived from plasma obtained
from blood donations, there is a risk of contamination of
the thrombin by any viruses present in the original blood
donation. Thus, any human thrombin preparation designed
for clinical use, should be subjected to a virus
inactivation step, prior to use.
Virus-inactivation by solvent-detergent treatment
has been described previously (13). However, the
thrombin preparation may need to be subjected to further
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W 096/26269 PCT/GB96/00423
purification steps in order to remove the solvent-
detergent. Other workers have described the use of
virus/inactivated prothrombin feedstocks, but have not
described methods for virus/inactivation of the thrombin
products prepared from them, for example EP 0378798 and
EP 0543178. Terminal (e.g. a final step of a process)
viral-inactivation of the product is viewed as probably
the safest and most effective method of
virus/inactivation, as it minimises the chance of
recont~in~tion.
There is thus a requirement in the art to produce
thrombin which is terminally virus/inactivated,
especially by heat-treatment.
Generally speaking the present invention is based on
the surprising discovery that prothrombin can be
converted to thrombin in good yield, under acidic
conditions and that these acidic conditions promote the
stability of the thrombin generated.
SummarY of the Invention
More specifically, a first aspect of the present
invention provides a process for preparing thrombin which
comprises treating a mixture comprising prothrombin,
Factor Xa, Factor Va and phospholipids with calcium ions
at a pH less than pH 7Ø
Generally, the mixture comprising prothrombin,
Factor Xa, Factor Va and phospholipids may be obtained
from a supernatant of a cryoprecipitate (which is formed
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W ~961~6~69 PCT/~D5~/00~23
by freezing and thawing plasma) of human plasma. The
mixture may be obtained by chromatographic purification
of the supernatant of cryoprecipitated plasma, generally
by anion-exchange chromatography. More particularly a
DEAE-cellulose eluate of absorbed supernatant of
cryoprecipitated plasma, which may be used for the
production of clinical Factor IX concentrates, can serve
as the mixture for thrombin production (14). The mixture
may comprise additional clotting factors, such as, Factor
X, Factor V, Factor IX, Factor IXa and trace amounts of
thrombin.
The prior art (e.g. EP 0378789 and EP 0528701) has
previously taught the addition of low levels of calcium
ions (5-25mM) to a mixture comprising prothrombin, at or
around physiological conditions (pH 7.0-7.3) and EP
0528701 describes that the addition of higher levels of
CaCl2 inhibits the preparation of thrombin. It might be
expected that conversion of prothrombin to thrombin would
proceed best in conditions which approach those of
physiological conditions. It is thus a surprising
feature of the present invention that thrombin may be
prepared in particularly good yield at a pH of less than
pH 7Ø Preferably the pH is between pH 6.0-7.0 and more
preferably between pH 6.4-6.6. Without wishing to be
restricted to any postulated theories, it is thought that
the pH of less than pH 7.0 limits autodegradation of
thrombin produced.
Generally the pH of less than pH 7.0 may be
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W 096t26269 PCT/GB96/00423
generated by the addition of Ca2+ ions, (in particular
CaCl2) at concentrations of 50mM-9OmM, more preferably
60mM-80mM and most preferably 6SmM-75mM, to the mixture.
Addition of CaCl2 in the ranges specified, further
generally results in a drop in the pH of the mixture
which may be sufficient to reach the required pH.
Alternatively, the mixture may be buffered to
between pH 6.0-7.0 or more preferably between pH 6.4-6.6
by any suitable buffer known to buffer in the required
range, before adding Ca 2+ ions to initiate the conversion
of prothrombin to thrombin. Examples of suitable buffers
include MES (2-[N-Morpholino]ethanesulphonic acid); ACES
(2-[2-Amino-2-oxoethyl)amino]ethanesolphonic acid); BES
(N,N-bis[2-Hydroxyethyl]-2-aminoethanesulphonic acid);
MOPS (3-[N-Morpholino]propanesulphonic acid); TES (N-
tris[Hydroxymethy]methyl-2-aminoethanesulphonic acid) and
HEPES (N-[2-Hydroyethyl]piperazine-N-[2-ethanesulphonic
acid) and the like.
In order to convert substantially all the
prothrombin to thrombin, the conversion should proceed
for a period of time and at a suitable temperature to
effect conversion. Typically the conversion should be
allowed to proceed for 12-24 hours and more preferably
for 16-20 hours. The conversion may proceed at room
temperature, typically between 18-25~C and does not
require incubation at higher temperatures.
Generally throm~in prepared in this manner has a
thrombin clotting activity of between 4,000-9,000 U/ml
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WO 96126269 PCT/G1~96/00423
and a specific activity of between 250-700 U/mg. This is
considerably higher than the activity of the thrombin
prepared by the process described in EP 0528701 (clotting
activity 700-1,000 U/ml and a specific activity of 20-40
u/mg)-
Some unwanted insoluble material may be found in thethrombin preparation probably due to the generation of
fibrin by the action of generated thrombin on any
fibrinogen present as a contaminant in the original DEAE-
cellulose eluate and of insoluble calcium phosphate. The
unwanted insoluble material may be removed by
centrifugation or by a filtering process. However, in
some instances, the preparation is too viscous and so the
thrombin preparation is preferably diluted to reduce the
viscosity. A dilution of 1 volume of thrombin
preparation with up to 3 volumes buffer, for example 3
volumes which can be any buffer suitable for use in the
range of pH 6.0-7.0, is generally carried out. Typical
buffers include 40mM sodium gluconate or 20mM MES, both
at pH 6.5. The diluted preparation may then be
centrifuged or filtered to remove any insoluble material.
Alternatively 20mM citrate, pH 6.5 may be used as the
diluting buffer. This may remove the need for
centrifugation or filtering, possibly due to the
solubilisation of insoluble calcium phosphate.
The diluted thrombin preparation is suitable for
immediate further processing, or may be stored at -400C
for at least six months without substantial loss of
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clotting activity. Alternatively, the diluted material
may be formulated and freeze-dried as an intermediate
purity preparation.
A specific activity of the thrombin preparation of
between 250 U/mg to 700 U/mg is equivalent to a thrombin
purity of between about 6%-17.5% based on a comparison to
a specific activity of pure ~-thrombin of 4,000 U/mg.
While this is sufficient in most clinical instances, it
is possible to subject the thrombin preparation to
further processing to yield a thrombin of higher purity.
Further processing may comprise chromatographic
purification of thrombin with an optional
solvent/detergent virus inactivation step prior to
chromatographic purification. A suitable
solvent/detergent virus/inactivation step has been
previously described by Edwards et al (13).
Chromatographic purification is generally carried
out by cation-exchange chromatography. Typical cation-
exchange resins which may be employed include Mono-S
(TRADEMARK), S-Sepharose FF (TRADEMARK) and S-Sepharose
Big Beads (TRADEMARK) although other sulphonate gels or
other cation-exchangers may be employed. The
chromatography step serves to remove solvent and
detergent, if a solvent/detergent virus inactivation step
has been carried out and also serves to purify the
thrombin preparation.
Typically the thrombin preparation is bound to the
cation-exchange chromatography resin and a purified
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WO 96126269 PCT/GB96~0042;~
thrombin is eluted using a suitable buffer with increased
salt concentration. Examples of suitable buffers include
20mM citrate pH 6.5, 20mM MES p~ 6.5 and 40mM gluconic
acid pH 6.5. The pH of the buffer should preferably be
in the range of pH 6.0-7.0 and more preferably pH 6.4-6.6
in order to preserve the activity of the purified
thrombin. Usually several salts are suitable for eluting
with any given cation-exchange resin and typically these
include NaCl.
The concentration of eluted purified thrombin
depends directly upon the amount bound to the resin, but
typically concentrations of purified thrombin between
4,000-9,000 U/ml may be obtained. Even the lower range
of these concentrations is adequate to allow suitable
dilution with a formulation buffer, for subsequent
freeze-drying.
Purified thrombin may be frozen directly in elution
buffer and stored for up to six months without
substantial loss of thrombin activity. However, for ease
of storage it is desirable that the intermediate purity
thrombin and purified thrombin, be freeze-dried.
Freeze-drying often results in a loss in activity of
thrombin (intermediate purity thrombin and purified
thrombin) and it is thus important to formulate the
thrombin with a formulation buffer. This formulation
buffer helps stabilise the thrombin during freeze-drying.
Prior to formulating the thrombin, it is often desirable
to centrifuge and/or filter the thrombin to remove
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insoluble material.
The art has previously described that the addition
of stabilising agents such as polyols, for instance,
glycerol, mannitol and sorbitol; sugars such as sucrose
and glucose and/or exogenous proteins such as albumin, to
a thrombin preparation is desirable to improve the
stability of a thrombin preparation, especially during
freeze-drying. It is thus a surprising feature of the
present invention that thrombin may be prepared, which is
substantially stable without additional stabilising
agents such as polyol, sugar, protein and mixtures
thereof.
Thus, in a further aspect the present invention
provides a thrombin preparation, the preparation
comprising thrombin substantially free of exogenous
stabilising agents (such as protein, sugar, polyol and
mixtures thereof) buffered at a pH of less than pH 7Ø
Generally the thrombin preparation is freeze-dried
and optionally heat-treated. Thus, in a still further
aspect, the present invention provides a freeze-dried,
optionally heat-treated, thrombin preparation,
substantially free of exogenous stabilising agents, such
as protein, sugar or polyol and mixtures thereof.
Preferably the thrombin preparation is buffered to
between pH 6.0-7.0, more preferably pH 6.4-6.6. This may
be achieved by for instance 40mM gluconic acid or 20mM
MES buffer in the suitable pH range. Preferably, the
thrombin preparation further comprises citrate at a
- ~ = ~
CA 02212832 1997-08-13
concentration of lOmM-30mM, typically sodium citrate.
More preferably the preparation further comprises sodium
chloride at a concentration of between 100-250m,M for
example 100-200mM. A thrombin preparation comprising
citrate and sodium chloride in addition to gluconate or
MES has been found to be most stable to freeze-drying and
optional heat-treatment. That is, the thrombin
preparation retains a greatest percentage of clotting
activity after freeze-drying and optional heat-treating.
Freeze-drying is preferably carried out employing a
two-stage freezing procedure. The frozen product is then
primary dried at a shelf temperature of -20~ to -30~C and
then secondary dried at a shelf temperature of +15~ to
+30~C.
The freeze-dried thrombin preparation may then be
heat-treated in order to inactivate any virus
contaminants. Typically dry heat-treatment is carried
out at temperatures of between 70~C to 100~C for up to 96
hours. A particularly preferred heat-treatment is
approximately 80~C for around 72 hours.
Detailed Description of Preferred Embodiments
Embodiments of the present invention will now be
described by way of Example, with reference to the
attached Figures.
t
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ExamPles Section
Example 1 - Preparation of a mixture comprisinq
Prothrombin Factor Xa Factor Va and phos~holipid by
DEAE-cellulose
450 litres of cryoprecipitate plasma was adjusted to
pH 6.9 + 0.05 and diluted with 150 litres of pyrogen free
H20 to a final volume of 600 litres. 6kg of DEAE-
cellulose gel (DE-52 Whatman) was then added to the
plasma/water solution and the resulting suspension mixed
continuously for one hour to bind the clotting factors to
the gel. The gel was then collected by centrifugation
and the supernatant discarded. The gel was then
resuspended in 30mM citrate, 30mM phosphate pH 6.9 buffer
and the resulting suspension was poured into a
chromatography column. The column was then packed by
washing with 21 litres of the same buffer. The clotting
Factors were then eluated from the column with an elution
buffer of 30mM citrate, 30mM phosphate, 200mM NaCl, pH
6.9. The eluate pool (3.1 litres) was then filtered
(0.45~m pore size) into sterile bottles and frozen.
The eluate pool contains substantial amounts of
prothrombin (Factor II) (at 80~M and about 25% of the
total protein). It also includes factors IX and X,
activated and non-activated (at about 5~M), coagulant-
active phospholipid and sufficient trace amounts of
Factors V and VIII to support the physiological
conversion of prothrombin to thrombin via the intrinsic
clotting pathway.
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Exam~le 2 - Preparation of intermediate Purity thrombin
Frozen DEAE-cellulose eluate (prepared according to
Example 1) was thawed at room temperature or in a 37~C
water bath. (Typical values of the eluate were as
follows: conductivity = 17mS; pH = 7.0; 30mM citrate;
30mM phosphate; 200mM sodium; 200mM chloride; 15 mg/ml
total protein and prothrombin 60 U/ml)
lM CaCl2 solution was then added dropwise to the
thawed eluate, with stirring, at a ratio of 75ml CaCl2 to
l,OOOml eluate, at 20OC. This resulted in a final
calcium concentration of 70mM and a drop in pH in the
mixture to pH 6.4-6.6. The reaction was allowed to
proceed with stirring overnight for 18 hours at 20~C, to
convert the prothrombin to thrombin.
In 15 experiments, the thrombin clotting activity
was 6,333 +1,146 U/ml (mean +SD) and specific activity of
508+ 110 U/mg (see Figure 1). SDS PAGE indicated that
effectively all the prothrombin band was converted into
bands co-migrating with thrombin, by the end of the
activation period.
Thrombin clotting activity was measured by
fibrinogen clotting time at room temperature with visual
detection and duplicate samples. To 200~1 of human
fibrinogen solution at 5 mg/ml in 50mM tris-HCl lOOmM
NaCl pH 7.5 was added lO0~1 of standard (1-4 U/ml) or
test solutions of thrombin diluted in 50mM tris-HCl,
lOOmM NaCl pH 7.5 supplemented with lOOmM CaCl2 and 0.1%
w/v bovine serum albumin, whereupon time to subse~uent
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clot formation was recorded. A standard curve was
constructed by plotting log~0 thrombin concentration
(U/ml) against log~0 clotting time (sec) using bovine
thrombin st~n~rdised against the human alpha-thrombin
standard 89/588. Thrombin clotting activity of test
samples was derived by extrapolation from the standard
curve (15).
ExamPle 3 - Effect of varvinq the PH of the reaction
solution during activation
Following the procedure described in Example l,
resulted in a mixture with a pH of 7.0-7.2. This
immediately decreased to pH 6.5 on addition of CaCl2 to
70mM. There was then a steady decrease in pH to 6.1-6.3
during the conversion period (18 hours). The fall in pH
was a requirement for the successful generation of
thrombin of high activity. This was demonstrated by
co~p~rative experiments, where the pH of the solution was
adjusted to pH 7.0 or pH 7.5 immediately after the
addition of CaCl2. Here the final pH values at the end of
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W 096l26269 PCTJGB96J0~423
the reaction period were pH 6.7 and pH 7.1 respectively
- and a much lower amount of thrombin activity was
generated (see Table 1).
~51
,lo p~ of tho p~ of t~o Clotti~g
~i~turo, ~ixturo activity
~ tely aftQr ~t the ond of (IU/~
CaCl2additio~ tho
(adjusted as c~v~.~ion
noco~sary) poriod
(18 hours)
1 6.5 6.1 5716
2 7.0 6.7 2012
3 7.5 7.1 1493
In a further experiment, the mixture was buffered
(20mM MES) to pH 6.5 immediately after CaCl2 addition.
This resulted in an additional small increase in
conversion to thrombin, but the increase in clotting
activity was insignificant.
Example 4 - Effects of varYinq the lenqth of time or
temperature emPloved for conversion of Prothrombin to
thrombin
Studies were carried out to determine the optimum
time course for the conversion of prothrombin to
thrombin. A comparison of the amount of thrombin
generated at 16 and 24 hours indicated that a plateau had
been reached by 16 hours.
.
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16
An investigation was also carried out to determine
the effect of incubation at 37~C for one hour prior to
subsequent room temperature incubation, in view of a
report that this step was necessary to obtain useful
yields with this type of feedstock (European Patent
Application No. 92401889.8). It was found that while the
initial rate of thrombin generation exceeded that
obtained at room temperature, the final yield of thrombin
was no better at 16 or 24 hours as compared to conversion
at room temperature.
Example 5 - Effects of varyinq calcium ion concentration
on thrombin Production
The amount of thrombin generated at 24 hours with a
range of added calcium ion concentrations (seven batches
of DEAE-cellulose eluates) was determined (Figure 2). It
was found that the addition of 70mM calcium consistently
resulted in efficient conversion of prothrombin to
thrombin.
ExamPle 6 - Viral inactivation by solvent/deterqent
Thrombin prepared according to Example 2 was mixed
by stirring with 0.3% tri-(n-butyl) phosphate and a 1%
solution of Tween 80 at a temperature of 20O-30~C for 6
to 24 hours. This was sufficient to inactivate any
contaminating lipid-enveloped viruses. The
solvent/detergent was removed by chromatography.
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Exam~le 7 - ChromatoqraPhic ~urification of intermediate
puritY thrombin
The chromatography step serves to remove solvent and
detergent and to purify the intermediate purity thrombin.
A 1.6cm diameter chromatography column was packed with
lOml of S-Sepharose FF (TRADEMARK) at a linear flow rate
of 2.2cm/min (equivalent to 4.5ml/min) using 40mM
gluconic acid, 20mM MES, or 20mM citrate all at pH 6.5.
lOOml of solvent/detergent treated thrombin according to
Example 6 or intermediate purity thrombin according to
Example 2, following a 1 + 3 dilution in equilibrating
buffer was filtered at 0.45~m and applied to the column
at the same flow rate. The column was then washed with
equilibrating buffer until the absorbency at 280nm
returned to baseline and solvent or detergent were
detectable only below acceptable low levels, in the
column effluent (typically approximately lSOml).
Thrombin was then eluted from the column by washing with
equilibrating buffer containing 0.5M NaCl. Purified
thrombin was obtained in about 25ml at a typical
concentration of 4,000 U/ml and 2mg/ml protein.
The typical yield of purified thrombin after the
chromatography step was 88+ 16%. This yield refers to a
thrombin preparation which was not subjected to a
- solvent/detergent virus inactivation step as described in
Example 6.
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ExamPle 8 - Formulation freeze-dryinq and terminal dr~
heat treatment
Thrombin prepared according to Examples 2 or 6 was
centrifuged at 3,000 rpm for 20 minutes at room
temperature and then filtered through a Millpore pre-
filter (AP25) followed by a Whatman 0.2~m filter
(Polydisc AS). The filtered solution was then diluted in
a formulation buffer (40mM gluconic acid or 20mM MES,
20mM trisodium citrate, 150mM NaCl, pH 6.5) to a thrombin
activity of 600 U/ml and dispensed in 2 ml lots into
glass vials for freeze-drying.
Freeze-drying was performed in a super-Modulyo
(Edwards, Crawley) freeze-dryer with a freezing
temperature setting of -45~C, followed by a primary
drying temperature setting of -25~C and a secondary
drying temperature setting of +20~C.
The vials were then heat-treated to inactivate any
cont~;n~ting viruses, at 80~C for 72 hours.
Exam~le 9 - Com~arison of a stabilisinq effect on
thrombin of various formulation buffers
Thrombin was formulated in a variety of formulation
buffers, in order to determine the optimum formulation
buffer for stabilising thrombin during freeze-drying and
subsequent heat-treatment (virus-inactivation).
Intermediate purity thrombin prepared according to
Example 2 and purified thrombin prepared according to
Example 7, were diluted with various formulation buffers
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(as described in Table 2) to a thrombin concentration of
600 U/ml. The thrombin preparation was then freeze-dried
according to Example 8 and a quantity of the freeze-dried
thrombin was also subjected to a heat-treatment of 80~C
for 72 hours. Thrombin clotting activity was determined,
as previously described, to determine the percentage
clotting activity that remained after freeze-drying and
subseguent heat-treatment. The results are shown in
Table 2.
It can be seen from Table 2 that formulation buffers
comprising 20mM tris-HCL buffer at pH 7.2 with or without
20mM trisodium citrate and/or 150mM sodium chloride,
resulted in a recovery of thrombin clotting activity,
after freeze-drying, of greater than 74%. However, large
losses in activity were seen post-heat-treatment,
particularly in the absence of trisodium citrate. The
inclusion of sodium chloride in the formulation buffer
gave rise to an intact plug of material, whereas without
sodium chloride, the plug retracted and collapsed.
Protein (e.g. Human albumin) can also be included in
the formulation at concentrations of 0.5g/l - lOg/l, to
act as a bulking agent and improve plug structure and '
appearance.
When the formulation buffer was made acidic by using
gluconic acid or MES buffered at pH 6.5, recovery of
thrombin clotting activity after dry heat-treatment was
substantially improved.
Long term stability was determined using the
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gluconic acid buffer formulation (see Table 2). These
studies were performed by storing several vials at 4~C
and 37~C, after freeze-drying and heat treatment. No
loss in thrombin clotting activity was observed over a
six month period, when comr~ring the 37~C stored thrombin
to the 4~C stored thrombin.
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~CABLE; 2
Recovery of clotting activity ~%)
tion Int~ t- purity High purity thrombin
~ufferthrombin
Post-FD Post-HT Post-FD Post-HT
20mM Tris-HCL
pH 7.2 96 12 93 12
20mM Tris-HCL
pH 7.2 + 20mM 92 56 91 56
trisodium
c~trate
20mM Tris-HCL
pH 7.2 + lSOmM 87 10 74 4
NaCl
20mM Tris-HCL
pH 7.2 + 20mM 91 41 90 51
trisodium
citrate
+ lSOmM NaCl
20mM gluconic
acid 100 99 93 85
+20mM
tri~o~i
citrate +
150mM
NaCl pH 6.5
20mM MES+ 20mM
trisodium nd 97 nd 86
citrate
+ 150mM NaCl
pH 6.5
FD = Freeze-drying HT = Heat-treatment of 80~C for 72 hours in
vial
nd = not done
- , , ;. . ," , . , , , - .
-,.. . - ., - . , ;, ..
r . ' ~ ~ ~
_ . ~ ,7 ,=
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REFERENCES
1. Fenton, J. W. (1981). Thrombin specificity. Ann. N.
Y. Acad. Sci. 370:468-495.
2. Suttie, J. W. and C. M. Jackson. (1977).
Prothrombin structure activation and biosynthesis.
Physiol. Rev. 57:1-65.
3. Brennan, M. (1991). Fibrin Glue. Blood Rev 5:240-
244.
4. Gibble, J. W. and P. M. Ness. (1990). Fibrin Glue -
The Perfect Operative Sealant. Transfusion 30:741-747.
5. Banninger, H., T. Hardegger, A. Tobler, A. Barth, P.
Schupbach, W. Reinhart, B. Lammle, and M. Furlan. (1993).
Fibrin Glue in the Surgery - Frequent Development of
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